Diethyl-[6-(4-hydroxycarbamoyl-phenyl-carbamoyloxy-methyl)-naphthalen-2-yl-methyl]-ammonium chloride for use in the treatment of muscular dystrophy

ABSTRACT

The present invention relates to a method for treating muscular dystrophy which comprises administering diethyl-[6-(4-hydroxycarbamoyl-phenyl-carbamoyloxy-methyl)-naphthalen-2-yl-methyl]-ammonium chloride or other pharmaceutically acceptable salts and/or solvates thereof to a patient in need of such a treatment. The invention further relates to a method for treating muscular dystrophy which comprises administering diethyl-[6-(4-hydroxycarbamoyl-phenyl-carbamoyloxy-methyl)-naphthalen-2-yl-methyl]-ammonium chloride or other pharmaceutically acceptable salts and/or solvates thereof in combination with one or more additional anti-inflammatory active agents to a patient in need of such a treatment.

This application is a continuation of U.S. patent application Ser. No.14/444,959, filed Jul. 28, 2014; which is a continuation ofPCT/IT2012/000040, filed Feb. 3, 2012; the contents of theabove-identified applications are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

The present invention relates to a method for treating musculardystrophy which comprises administeringdiethyl-[6-(4-hydroxycarbamoyl-phenyl-carbamoyloxy-methyl)-naphthalen-2-yl-methyl]-ammoniumchloride or other pharmaceutically acceptable salts and/or solvatesthereof to a patient in need of such a treatment.

The invention further relates to a method for treating musculardystrophy which comprises administeringdiethyl-[6-(4-hydroxycarbamoyl-phenyl-carbamoyloxy-methyl)-naphthalen-2-yl-methyl]-ammoniumchloride or other pharmaceutically acceptable salts and/or solvatesthereof in combination with one or more additional anti-inflammatoryactive agents to a patient in need of such a treatment.

BACKGROUND OF THE INVENTION

Muscular dystrophies (MDs) include a heterogeneous group of geneticdiseases invariably leading to muscle degeneration and impairedfunction. Mutation of nearly 30 genes gives rise to various forms ofmuscular dystrophy, which differ in age of onset, severity, and musclegroups affected (Dalkilic I, Kunkel LM. (2003) Muscular dystrophies:genes to pathogenesis. Curr. Opin. Genet. Dev. 13:231-238).

The most common MD is the Duchenne muscular dystrophy (DMD), a severerecessive X-linked disease which affects one in 3,500 males,characterized by rapid progression of muscle degeneration, eventuallyleading to loss of ambulation and death within the second decade oflife.

Attempts to replace or correct the mutated gene, by means of gene orcell therapy, might result in a definitive solution for musculardystrophy, but this is not easy to achieve. Alternative strategies thatprevent or delay muscle degeneration, reduce inflammation or promotemuscle metabolism or regeneration might all benefit patients and, in thefuture, synergize with gene or cell therapy. Steroids that reduceinflammation are currently the only therapeutic tool used in themajority of DMD patients (Cossu G, Sampaolesi M. (2007) New therapiesfor Duchenne muscular dystrophy: challenges, prospects and clinicaltrials. TRENDS Mol. Med. 13:520-526).Diethyl-[6-(4-hydroxycarbamoyl-phenyl-carbamoyloxy-methyl)-naphthalen-2-yl-methyl]-ammoniumchloride, which is described in WO 97/43251 (anhydrous form) and in WO2004/065355 (monohydrate crystal form), herein both incorporated byreference, is an anti-inflammatory agent which is able to inhibit thesynthesis of the majority of pro-inflammatory cytokines whilst sparinganti-inflammatory ones.

Diethyl-[6-(4-hydroxycarbamoyl-phenyl-carbamoyloxy-methyl)-naphthalen-2-yl-methyl]-ammoniumchloride is also known as ITF2357.

The monohydrate crystal form ofdiethyl-[6-(4-hydroxycarbamoyl-phenyl-carbamoyloxy-methyl)-naphthalen-2-yl-methyl]-ammoniumchloride is known as Givinostat.

Givinostat is being evaluated in several clinical studies, includingstudies for the treatment of myeloproliferative diseases, polycythemiavera, periodic fever syndrome, Crohn's disease and systemic-onsetjuvenile idiopathic arthritis. Orphan drug designation was assigned inthe E.U. for the treatment of systemic-onset juvenile idiopathicarthritis and for the treatment of polycythemia vera.

Givinostat has been recently found to act also as a Histone Deacetylaseinhibitor (WO 2011/048514).

Histone deacetylases (HDAC) are a family of enzymes capable of removingthe acetyl group bound to the lysine residues in the N-terminal portionof histones or in other proteins. HDACs can be subdivided into fourclasses, on the basis of structural homologies. Class I HDACs (HDAC 1,2, 3 and 8) are similar to the RPD3 yeast protein and are located in thecell nucleus. Class II HDACs (HDAC 4, 5, 6, 7, 9 and 10) are similar tothe HDA1 yeast protein and are located both in the nucleus and in thecytoplasm. Class III HDACs are a structurally distinct form ofNAD-dependent enzymes correlated with the SIR2 yeast protein. Class IV(HDAC 11) consists at the moment of a single enzyme having particularstructural characteristics. The HDACs of classes I, II and IV are zincenzymes and can be inhibited by various classes of molecule: hydroxamicacid derivatives, cyclic tetrapeptides, short-chain fatty acids,aminobenzamides, derivatives of electrophilic ketones, and the like.Class III HDACs are not inhibited by hydroxamic acids, and theirinhibitors have structural characteristics different from those of theother classes.

The expression “histone deacetylase inhibitor” in relation to thepresent invention is to be understood as meaning any molecule ofnatural, recombinant or synthetic origin capable of inhibiting theactivity of at least one of the enzymes classified as histonedeacetylases of class I, class II or class IV.

Although HDAC inhibitors, as a class, are considered to be potentiallyuseful as anti-tumor agents, it is worth to note that, till now, onlytwo of them (Vorinostat and Romidepsin) have been approved as drugs forthe cure of a single tumor form (Cutaneous T-cell lymphoma).

It is evident that the pharmaceutical properties of each HDAC inhibitormay be different and depend on the specific profile of inhibitorypotency, relative to the diverse iso-enzymes as well as on theparticular pharmacokinetic behaviour and tissue distribution.

Some HDAC inhibitors have been claimed to be potentially useful, incombination with other agents, for the treatment of DMD (WO 2003/033678,WO 2004/050076, Consalvi S. et al. Histone Deacetylase Inhibitors in theTreatment of Muscular Dystrophies: Epigenetic Drugs for GeneticDiseases. (2011) Mol. Med. 17:457-465).

The potential therapeutic use of HDAC inhibitors in DMD may however behampered by the possible harmful effects of these relatively toxicagents, especially when used for long-term therapies in paediatricpatients.

Givinostat, as anti-inflammatory agent, has been already used in a phaseII study in children with Systemic Onset Juvenile Idiopathic Arthritis;Givinostat administered at 1.5 mg/kg/day for twelve weeks achieved ACRPedi 30, 50 and 70 improvement of approximately 70% (Vojinovic J,Nemanja D. (2011) HDAC Inhibition in Rheumatoid Arthritis and JuvenileIdiopathic Arthritis. Mol. Med 17:397-403) showing only a limited numberof mild or moderate but short lasting, adverse effects.

To date more than 500 patients (including 29 children) have been treatedwith Givinostat. Repeated dose toxicity studies were carried out indogs, rats and monkeys. Oral daily doses of the drug were administeredup to nine consecutive months. The drug was well tolerated with no overttoxicity at high doses. The “no adverse effect levels” (NOAEL) rangedfrom 10 to 25 mg/kg/day depending on the animal species and the durationof treatment.

In juvenile animals Givinostat at 60 mg/kg/day did not affect thebehavioural and physical development and reproductive performance ofpups.

No genotoxic effect was detected for Givinostat in the mouse lymphomaassay and the chromosomal aberration assay in vitro and in themicronucleus test and UDS test in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows time to exhaustion in control group, TSA-treated group, andITF-treated group.

FIG. 2 shows that ITF treated group had an increased muscle sizecompared to the CTR group: (A) whole naked bodies, and (B) hind limblegs isolated from mdx.

FIG. 3 shows the analysis of “tibialis anterior” (TA) section area byMasson's trichrome staining in control group, TSA-treated group, andITF-treated group.

DETAILED DESCRIPTION OF THE INVENTION

We have now found that the administration ofdiethyl-[6-(4-hydroxycarbamoyl-phenyl-carbamoyloxy-methyl)-naphthalen-2-yl-methyl]-ammoniumchloride is able to prevent or delay muscle degeneration, reduceinflammation and promote muscle metabolism or regeneration.

An object of the present invention is therefore a method for treatingmuscular dystrophy, which comprises administeringdiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride, preferably in monohydrate form, more preferably in monohydratecrystal form, or other pharmaceutically acceptable salts and/or solvatesthereof, to a patient in need of such treatment.

The muscular dystrophy is, preferably, the Duchenne muscular dystrophy.

Diethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride, preferably in monohydrate form, more preferably in monohydratecrystal form, or other pharmaceutically acceptable salts and/or solvatesthereof of the invention is administered in an amount ranging from 0.5to 15 mg/kg/day, preferably from 1 to 10 mg/kg/day. More preferably, itis administered on a daily basis.

For the purpose of the present inventiondiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-ylmethyl]-ammonium chloride (or other pharmaceutically acceptable saltsand/or solvates thereof) is administered in the form of a pharmaceuticalcomposition containing the same together with at least onephysiologically acceptable excipient.

Said pharmaceutical composition is preferably administered to thepatient by enteral and/or parenteral route, preferably by oral,sublingual, rectal, intravascular, intravenous, subcutaneous route, morepreferably by oral route.

The pharmaceutical composition can be formulated in a solid or a liquidform. Preferably, said solid form is selected from tablet, granulate,aggregate, compressed or coated pill, hard or gelatine capsule and saidliquid form is selected from suspension or syrup.

The pharmaceutical composition of the invention preferably contains from7.5 to 200 mg ofdiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride or other pharmaceutically acceptable salts and/or solvatesthereof, more preferably from 25 to 150 mg, per unit dosage form.

For the purposes of the present invention,diethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride or other pharmaceutically acceptable salts and/or solvatesthereof can be effectively administered alone or it can be administeredin combination with at least one additional anti-inflammatory activeagent.

According to a preferred embodiment of the invention, said “at least oneadditional anti-inflammatory active agent” is a steroid.

The steroid is preferably a glucocorticoid corticosteroid, morepreferably it is selected from the group consisting of prednisolone,prednisone, deflazacort, hydrocortisone, methylprednisolone,dexamethasone, betamethasone, triamcinolone, beclometasone,fludrocortisone acetate, deoxycorticosterone acetate, even morepreferably it is selected from prednisolone and deflazacort.

The combination therapy according to the present invention includesadministration of a single pharmaceutical dosage formulation comprisingdiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-ylmethyl]-ammonium chloride or other pharmaceutically acceptable saltsand/or solvates thereof and the at least one additionalanti-inflammatory active agent, as well as the administration ofdiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-ylmethyl]-ammonium chloride or other pharmaceutically acceptable saltsand/or solvates thereof and the at least one additionalanti-inflammatory active agent each in their own separate pharmaceuticaldosage formulations.

When a single dosage formulation is used, it may contain from 7.5 to 200mg ofdiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride or the pharmaceutically acceptable salts and/or solvatesthereof, preferably from 25 to 150, and from 0.25 to 2.5 mg/kg of saidat least one anti-inflammatory active agent, preferably from 0.5 to 1mg/kg.

Where separate dosage formulations are used,diethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-ylmethyl]-ammonium chloride or other pharmaceutically acceptable saltsand/or solvates thereof and the at least one additional active agent canbe administered at essentially the same time, i.e., concurrently; or atseparately staggered times, i.e., sequentially. The combination therapyaccording to the present invention is understood to include all theseregimens.

According to one embodiment of the present invention, the recipient ofthe claimed method is a child.

In more detail,diethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride or other pharmaceutically acceptable salts and/or solvatesthereof, either alone or in combination with at least one additionalactive agent or the pharmaceutical composition thereof is administeredto a child, preferably on a daily basis, in an amount ranging from 0.5to 15 mg/kg/day, preferably from 1.0 to 10 mg/kg/day.

The following examples are intended to be illustrative of the inventionrather than limiting the scope thereof.

EXAMPLES Introduction

The animal model chosen for the experiment was the C57BL/10ScSn-Dmdmdx/Jmouse (hereafter “mdx”). The mdx mouse, an X-linked myopathic mutant, isthe most amenable and approachable disease model for preclinical studieson human Duchenne muscular dystrophy. The mdx mouse has a single basesubstitution within an exon, which causes premature termination of thepolypeptide chain. This mutation is X chromosome-linked and producesviable homozygous animals that lack the muscle protein dystrophin, havehigh serum levels of muscle enzymes, and possess histological lesionssimilar to human muscular dystrophy. The histological features, linkage,and map position of mdx make these mice a worthy animal model ofDuchenne muscular dystrophy. Mdx mice have a progressive muscledegeneration starting about three weeks of age. At eight weeks, the mdxforelimb strength decreases while the hind limbs show normal strength.These findings reflect the progression of the acute phase of musclenecrosis in the young mdx mice (Grounds et al., 2008). Muscle atrophy,inflammation and fibrosis are present in the mdx mice at eight weeks ofage (Consalvi S. et al. Histone Deacetylase Inhibitors in the Treatmentof Muscular Dystrophies: Epigenetic Drugs for Genetic Diseases. (2011)Mol. Med. 17:457-465).

Experiment Description:

2 months old mdx mice were randomized in three groups of ten animalseach.

-   -   Control group (CTR) was treated with a solution of        methylcellulose (0.5%, gavage) once a day for 90 days.    -   Test group (ITF) was treated with a        diethyl-[6-(4-hydroxycarbamoyl-phenyl-carbamoyloxy-methyl)-naphthalen-2-yl-methyl]-ammonium        chloride (ITF2357) solution (10 mg/Kg, gavage) once a day for 90        days.    -   Trichostatin A group (TSA) was treated with a TSA solution (0.6        mg/Kg, i.p.) once a day for 90 days.

Example 1 Givinostat Increases Resistance to Fatigue in Mdx Mice after90 Days Treatment (Treadmill Test)

The effect of the treatments was investigated on exercise performance invivo by the treadmill test, according to the following protocol:

Run: 8 cm/sec for 5 minAfter 5 min—progressive increase of 2 cm/minNumb: 0.2 mA (stable)End of run if stop more than 5-10 sec on plate.

As shown in FIG. 1, ITF treated group had a longer time to exhaustionthan the CTR group.

Example 2 Givinostat Treatment Increases the Size of Muscles in Mdx Mice(Macroscopic Evaluation)

At the end of the experiment described above, the mice were sacrificedfor skeletal muscle analysis by macroscopic evaluation.

As shown in FIG. 2, A) (whole naked bodies) and B) (hind limb legsisolated from mdx), ITF treated group had an increased muscle sizecompared to the CTR group.

Example 3 Givinostat Reduces the Fibrosis Area in Treated Mdx Mice(Masson's Trichrome Staining)

At the end of the treatment period, analysis of “tibialis anterior” (TA)section area by Masson's trichrome staining, revealed a strong reductionof fibrotic (blue) area for ITF treated group as shown in FIG. 3.

What is claimed is:
 1. A method for treating muscular dystrophy in apatient, comprising administering to a patient in need thereofdiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride, or the pharmaceutically acceptable salts and/or solvatesthereof, and at least one additional anti-inflammatory agent.
 2. Themethod according to claim 1, wherein said additional anti-inflammatoryagent is a steroid.
 3. The method according to claim 2, wherein saidsteroid is selected from the group consisting of prednisolone,prednisone, deflazacort, hydrocortisone, methylprednisolone,dexamethasone, betamethasone, triamcinolone, beclometasone,fludrocortisone acetate, and deoxycorticosterone acetate.
 4. The methodaccording to claim 1, wherein saiddiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride, or the pharmaceutically acceptable salts and/or solvatesthereof, and said additional anti-inflammatory agent are administeredsimultaneously, separately, or sequentially.
 5. The method according toclaim 1, wherein said muscular dystrophy is Duchenne muscular dystrophy.6. The method according to claim 1, wherein the administration to thepatient is on a daily basis.
 7. The method according to claim 1, whereinsaiddiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride, or the pharmaceutically acceptable salts and/or solvatesthereof is administered in an amount ranging from 0.5 to 15 mg/kg/day.8. The method according to claim 1, wherein said patient is a child. 9.The method according to claim 8, wherein saiddiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride, or the pharmaceutically acceptable salts and/or solvatesthereof is administered in an amount ranging from 1 to 10 mg/kg/day. 10.The method according to claim 1, wherein saiddiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride, or the pharmaceutically acceptable salts and/or solvatesthereof is administered in the form of a pharmaceutical compositioncomprising the same together with at least one physiologicallyacceptable excipient.
 11. The method according to claim 10, wherein saidpharmaceutical composition is administered by oral, sublingual, rectal,intravascular, intravenous, or subcutaneous route.
 12. The methodaccording to claim 11, wherein said pharmaceutical composition is in asolid or a liquid form.
 13. The method according to claim 12, whereinsaid solid form is selected from the group consisting of powder, tablet,granulate, aggregate, compressed pill, coated pill, hard gelatincapsule, and gelatin capsule.
 14. The method according to claim 12,wherein said liquid form is a suspension or a syrup.
 15. The methodaccording to claim 1, wherein saiddiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride is in a monohydrate form.
 16. The method according to claim 1,wherein saiddiethyl-[6-(4-hydroxycarbamoyl-phenylcarbamoyloxymethyl)-naphthalen-2-yl-methyl]-ammoniumchloride is in a crystal form.